Differentially methylated CpG regions analyzed by PCR-high resolution melting for monozygotic twin pair discrimination.

Discrimination between monozygotic (MZ) twins is a forensic limitation when using conventional DNA profiling techniques for human identification. Recent works based on epigenetics seem to open a new way to solve this issue due to methylation status of MZ twins change during their lifetime. Methylation analysis through BeadChip platforms allows the study up to 850 K CpG sites revealing that numerous differential methylation regions exist between MZ twins. However, this methodology is difficult to implement in forensic laboratories. On the contrary, PCR-HRM (High Resolution Melting) technology is one of the easiest methods for analyzing DNA methylation and it has been capable to discriminate between MZ twins. The purpose of this study is to contribute with new differential methylation regions in MZ twins to those that have been previously studied through PCR-HRM. Here, we have selected 6 CpG regions located at the ITGA2B, ASPA, PDE4C, ZIC5, USP11 and NOP14 loci that have shown methylation status variation during lifetime. The study has been carried out from saliva-derived DNA of 18 MZ twin pairs. The most discriminating regions were those located at ITGA2B, ASPA and ZIC5 loci showing significant within-pair differences in 44.4% of the cases. Non evidences of relation between age and significant differences between MZ twins were found, although the 50% of MZ twin pairs were discrimnated in the oldest age range (59-66 years old). These results support the use of these regions to increase the number of epigenetics age-related markers available to discriminate between MZ twins in a pair by PCR-HRM in forensic laboratories.